Pediatr Nephrol (2008) 23:55–61 DOI 10.1007/s00467-007-0641-9 ORIGINAL ARTICLE Vitamin D deficiency during pregnancy and lactation stimulates nephrogenesis in rat offspring Noori Maka & John Makrakis & Helena C. Parkington & Marianne Tare & Ruth Morley & M. Jane Black Received: 19 January 2007 / Revised: 22 July 2007 / Accepted: 6 August 2007 / Published online: 27 October 2007 # IPNA 2007 Abstract There is increasing evidence of vitamin D insufficiency in women of child-bearing age and their infants. This study examined the effect of maternal vitamin D deficiency on nephron endowment in rat offspring (n=7 per group). Sprague–Dawley dams were fed either a vitamin D deplete diet or a vitamin replete (control) diet prior to pregnancy, during pregnancy and throughout lactation. At 4 weeks of age the offspring were weaned and maintained on their respective diets until they were killed at 7 weeks. In the fixed right kidney, kidney volume, renal corpuscle volume and nephron number were stereologically determined. There was no difference between groups in body weight, kidney weight or kidney volume. There was a significant 20% increase in nephron number in kidneys of vitamin D deplete offspring (vitamin D deficient, 29,000± 1,858, control, 23,330±1,828; P=0.04). This was accompanied by a significant decrease in renal corpuscle size in the vitamin D deplete group compared with the controls (6.125±0.576×10−4 mm3 and 8.178±0.247×10−4 mm3, respectively; P=0.03). We concluded that maternal vitamin D deficiency in rats appears to stimulate nephrogenesis. Whether this confers a renal functional advantage or not is unknown. N. Maka : J. Makrakis : M. J. Black (*) Department of Anatomy & Cell Biology, Monash University, Post Office Box 13C, Clayton, Victoria 3800, Australia e-mail: jane.black@med.monash.edu.au H. C. Parkington : M. Tare Department of Physiology, Monash University, Clayton, Victoria 3800, Australia R. Morley Department of Paediatrics and Murdoch Children’s Research Institute, University of Melbourne, Parkville, Victoria 3052, Australia Keywords Vitamin D deficiency . Vitamin D insufficiency . Nephron . Nephrogenesis . Stereology . Nephron endowment . Renal development Introduction Vitamin D is an essential fat-soluble vitamin which is predominantly synthesised in humans by the action of sunlight (ultraviolet-B radiation) on the skin, with a small contribution from dietary intake [1]. There has been a recent increase in reports of vitamin D insufficiency in developed countries, and this has been attributed to genetic, social and environmental factors [2]. For example, the migration of populations with dark skins away from the Equator, extensive covering when outdoors (for personal, religious or cultural reasons), low dietary or supplementary vitamin D intake and an ageing population all contribute to an increased prevalence of vitamin D insufficiency [2]. In countries around the world, emphasis has been placed on the reduction of exposure to ultraviolet light to minimise skin cancer risk, concomitantly increasing the risk of vitamin D insufficiency [3]. Of particular concern is the rise in the incidence of vitamin D insufficiency in women of child-bearing age [4–7], and this brings into question the potential effects of prenatal vitamin D deficiency on the foetus and the implications for foetal programming [8]. Programming for later disease, in response to an adverse environment during early development, was initially proposed by Barker and colleagues in the late 1980s [9, 10], and is increasingly recognised as a likely predisposing factor in the pathogenesis of a number of clinical conditions [11–13]. Adaptations to an adverse environment during early development may confer short-term advantage but result in long-term vulnerability to disease [14]. DO00641; No of Pages 56 In this regard, it is important for us to gain an understanding of how maternal vitamin D deficiency during pregnancy affects renal development, since nephrogenesis in the term infant only occurs in foetal development (predominantly in late gestation) [15]. In the human, nephrogenesis is complete at 36 weeks’ gestation, with no new nephrons formed thereafter for the life of that individual [15]. In the rat, nephrogenesis continues after birth and ceases at approximately postnatal day 10 [16]. Perturbations in nephrogenesis are potentially important to adult renal health, since several studies have suggested that a reduced nephron endowment may be an important factor in the pathogenesis of hypertension and renal disease in adulthood [17–19]. Importantly, in animal studies, perturbations in utero have resulted in a congenital nephron deficit and have been linked to the prenatal programming of adult hypertension in some [20–22] (but not all [23, 24]) animal models and to susceptibility to renal disease [25]. Hence, given the importance of nephron endowment at birth and the recent resurgence of vitamin D deficiency in women of child-bearing age, it is imperative that we gain an understanding of how maternal vitamin D deficiency affects nephrogenesis in the offspring. In the present study we hypothesised that vitamin D deficiency in utero and in early postnatal life in the rat would lead to altered kidney development. Therefore, the aim of the present study was to determine the effect of maternal vitamin D deficiency, during pregnancy and lactation, on nephron endowment in rat offspring. Methods Animals and diet treatment Four-week-old female Sprague–Dawley rats were obtained from Monash Animal Services and divided into two experimental groups. One group was fed a vitamin Ddeficient, semi-purified, diet (AIN93G; Glenn Forrest Specialty Feeds, WA, Australia) without the inclusion of cholecalciferol (vitamin D3), whilst the second group received the same diet (AIN93G including 1,000 IU/kg of vitamin D3). Both diets contained 4.5 g/kg of calcium. Food and water was provided ad libitum. Six weeks later, the rat dams were mated with vitamin D-replete males, and the dams were maintained on the diets during pregnancy and lactation. At 4 days of age the litters were reduced to ten pups, and all experimental offspring weighed. At 23 days of age all offspring were again weighed and then weaned at 4 weeks of age. The offspring were maintained on their respective diets until 7 weeks of age, in facilities with a 12 h day/night cycle of incandescent (ultraviolet-B free) light. At 7 weeks of age the rats were Pediatr Nephrol (2008) 23:55–61 weighed, and a blood sample was collected via the tail vein. The rats were then euthanised, and, at necropsy, the right kidney was excised, weighed and immersion-fixed in 10% buffered formalin. Seven kidneys per group (from all litters) were randomly selected for stereological analyses. Kidneys from both male and female offspring were used, since previous studies have shown there to be no difference in nephron endowment between sexes [20, 26]. The animal experiments were approved by the Monash University, Biochemistry, Anatomy and Microbiology Animal Ethics Committee, and the treatment and care of the animals conformed with the Australian code of practice for the care and use of animals for scientific purposes. Measurement of serum 25-hydroxyvitamin D and calcium concentrations Serum was stored at −70°C for later determination of 25hydroxyvitamin D [25-(OH)D] and total serum calcium levels. Assays were undertaken in fully accredited laboratories at The Royal Children’s Hospital, Melbourne, that participate in national and international quality assurance schemes. 25-(OH)D was measured by radio-immunoassay (Immunodiagnostic Systems, Tyne and Wear, UK), an assay measuring 100% of 25-(OH)D3, with reportedly 75% crossreactivity with 25-(OH)D2. The coefficient of variation was 10.2% at 30 nmol/l and 10.1% at 100 nmol/l. Total calcium was measured with the Vitros 250 autoanalyser (OrthoClinical Diagnostics, NY, USA). Stereological estimation of kidney volume, nephron number and renal corpuscle volume Kidney volume The right kidney was cleaned of fat and connective tissue, weighed, and sliced into 1 mm slices with a razor blade slicing device. Every second kidney slice was embedded in glycol methacrylate and sectioned at 20 μm, with every 10th and 11th sections collected (beginning with a random number) and stained with haematoxylin and eosin. Every 10th section was projected onto a microfiche monitor; an orthogonal grid was superimposed, and the number of grid points overlying all kidney sections (Ps), as well as the number of grid points overlying complete kidney sections (Pf), were counted. The volume of the kidney was then determined by the Cavalieri principle [27, 28]: Vkid ¼ 10  t  aðpÞmicrofiche  Ps where 10 is the inverse of the section sampling fraction, t is the mean section thickness, a(p) is the area associated with each grid point and Ps is the total number of grid points counted. Pediatr Nephrol (2008) 23:55–61 Nephron number Using an unbiased physical disector/ fractionator stereological technique, we estimated the total number of glomeruli (and, thereby, nephrons) in the kidney in the sampled glycol methacrylate sections, as previously described [28, 29]. In brief, the intact sampled kidney sections (used in the estimation of Pf) were projected onto a tabletop by a microscope with a projection arm. The corresponding look-up (11th) section was also projected onto the table top, alongside the image of the sampled (10th) section. The fields of view between both sections were aligned, and an unbiased counting frame was superimposed over the image of the sampled section. The kidney sections were sampled by uniform systematic sampling. A physical disector approach was employed to count the number of glomeruli (Q−) within the sampled fields of view. We then estimated the total number of glomeruli within the kidneys by multiplying the Q− by the inverse of the sampling fractions, such that the total number of glomeruli was estimated according to the following equation [28, 30]: Nglom;kid ¼ 10  ðPs =Pf Þ  ð1=2f a Þ  Q where 10 is the inverse of the sections sampled, Ps/Pf is the inverse of the fraction of the tissue analysed in the sampled sections and 1/2 fa is the inverse of the fraction of the section area used to count the glomeruli. We calculated the numerical density (NVglom,kid, the number of glomeruli per volume of kidney tissue) by dividing the total number of glomeruli in the kidney (Nglom,kid) by the volume of the kidney (Vkid). Renal corpuscle volume In the glycol methacrylate sections, renal corpuscle volume was also stereologically measured [25]. The renal corpuscle is composed of Bowman’s capsule, Bowman’s space and the glomerular tuft. To measure this, an orthogonal grid was superimposed over the projected image of the sampled sections. The number of grid points falling on kidney tissue (Pkid) and on renal corpuscles (Pcorp) in the sampled area were counted. We determined the volume density of the renal corpuscle (VVcorp,kid) by dividing the number of points overlying renal corpuscles by the number of points overlying the kidney [28, 30]:  VVcorp;kid ¼ Pcorp Pkid We subsequently calculated the mean renal corpuscle volume by dividing the renal corpuscle volume density by the numerical density of glomeruli in the kidney [28, 30]:  Vcorp ¼ VVcorp;kid NVglom;kid 57 Statistical analysis The mean outcomes were compared between the vitamin Ddeficient group and the control group. For all data, gender was included in an initial analysis of variance (ANOVA) model to identify gender differences. Where gender was not significant, male and female data were pooled, and the data between groups were compared. Within each experimental group we performed a linear regression analysis to examine the relationship between kidney weight and nephron number. Data in the text are represented as means ± standard errors of the means. Results Serum 25-(OH)D and calcium concentrations In the 7-week-old rats the mean serum 25-(OH)D concentrations in the vitamin D deficient group were markedly reduced (P=0.01) in comparison with those of the control offspring (10.99 ± 1.670 nmol/l and 133.0±40.61 nmol/l, respectively), whereas the serum total calcium concentrations were not significantly different (2.256±0.257 nmol/l and 2.307±0.194 nmol/l, respectively). Body weights, kidney weights and kidney volumes In the rats at postnatal day 4 there was no significant difference in body weights of the offspring (pooled data of males and females) in the control and vitamin D-deficient groups (19.9±0.9 g and 19.8±2.6 g, respectively). Likewise, in the 23-day-old rats, there was no difference in body weights of the offspring (male and female combined) between groups, with body weights in controls averaging 60.9±1.2 g and those in the vitamin D-deficient offspring averaging 60.9±6.7 g. At 7 weeks of age, female offspring were significantly lighter (P=0.002) than males; however, there was no significant effect of vitamin D deficiency on body weight. The effect of gender on body weight did not differ between the two experimental groups (Table 1). Likewise, there was no significant difference in kidney weight or in kidney weight to body weight ratio between the two experimental groups (Table 1). In accordance with the kidney weight data there was no significant difference in kidney volume between the vitamin D-deficient group and the control group (Fig. 1). Kidney weight and kidney volume were not affected by gender, whereas the kidney weight to body weight ratio was significantly higher (P=0.019) in females. 58 Pediatr Nephrol (2008) 23:55–61 Table 1 Body weight, kidney weight, kidney weight to body weight ratio and numerical density of glomeruli in control (n=7) and vitamin D-deficient (n=7) offspring at 7 weeks of age Parameter Control Vitamin D-deficient Body weight (g) Kidney weight (mg) Kidney weight:body weight (mg:g) Numerical density (number of glomeruli per cubic millimetre) 196.4±39.2 815±137 4.22±0.26 39.1±1.9 202.6±46.9 844±140 4.24±0.26 48.2±4.2 Renal morphology Discussion There was an observable increased density of glomeruli of reduced size in the kidneys from the vitamin D-deficient group, but no other morphological abnormalities were observed in the cortex, medulla or papillae (Fig. 2). In this study, vitamin D deficiency from conception to 7 weeks postnatally in rats did not affect kidney size, but, importantly, it led to a 20% increase in nephron endowment, independent of kidney size, with a concomitant reduction in renal corpuscle size. Further studies are required, firstly to elucidate whether the maturation of the nephrons (in particular, glomerulogenesis) in the vitamin D-deficient offspring is normal and, secondly, to establish whether nephron and/or renal function are normal. Vitamin D plays a key role in calcium homoeostasis and bone metabolism [1, 31]. In this study, serum total calcium Nephron number Although there was no difference in the sizes of the kidneys between the vitamin D-deficient and control groups, there was a 20% increase in nephron number in the kidneys of animals from the vitamin D-deficient group (P=0.04) (Fig. 3a). There was no effect of gender on nephron number or numerical density of glomeruli. Importantly, linear regression analysis demonstrated a significant linear correlation between kidney weight and nephron number in the control offspring (R2 =0.668; P=0.025), but this relationship was not observed in the vitamin D-deficient offspring (R2 =0.006; P=0.871) There was a trend for the numerical density (number of glomeruli per cubic millimetre of kidney tissue) to be increased, with much increased variation in size, in vitamin Ddeficient animals than in controls (Table 1), which is reflected in the absence of statistical significance. Renal corpuscle size There was a significant decrease (P=0.03) in the average size of the renal corpuscle in the kidneys from the vitamin Ddeficient rats compared with that in controls, as demonstrated by a significant decrease in renal corpuscle volume (Fig. 3b). Gender had no effect on renal corpuscle volume. Kidney volume (mm3) 1000 750 500 250 0 +Vit D -Vit D Fig. 1 Volumes of right kidneys in rats at 7 weeks of age in control (+Vit D) and vitamin D-deficient (−Vit D) offspring. Data are expressed as mean ± standard error of the mean (SEM) Fig. 2 Representative light micrographs of the renal cortex in (a) control and (b) vitamin D-deficient kidneys. There was an observable increase in glomerular density in the renal cortex in the vitamin D deficient group compared with that in the control group (×240) Pediatr Nephrol (2008) 23:55–61 59 a Nephron Number 40000 30000 20000 10000 0 +Vit D -Vit D Renal Corpuscle Volume (x 10-4 mm3) b 10.0 7.5 5.0 2.5 0.0 +Vit D -Vit D Fig. 3 Stereological estimates of nephron number (a) and renal corpuscle volume (b) in the rats at 7 weeks of age in the right kidneys of control (+Vit D) and vitamin D-deficient (−Vit D) offspring. Data are expressed as mean ± standard error of the mean (SEM) concentration did not differ between the deficient and control groups, even though serum 25-(OH)D concentrations were markedly different. It is important to note that, in the present study, total calcium was measured and not free calcium, which is the biologically active component. Total calcium, as measured in this study, includes both free and inactive albumin-bound calcium. Interestingly, our findings are analogous to those recently described in pregnant women, where total calcium concentration was not related to that of 25-(OH)D [32]. The mechanisms for the maintenance of the serum total calcium concentration in the presence of low circulating 25-(OH)D concentration could not be determined in our study. In primates, nephron endowment in the kidney correlates with birth weight and kidney size [33, 34]. In rat studies, where nephrogenesis continues after birth, such correlations are often observed in the offspring at weaning [23]. Surprisingly, in our study, there was an increase in nephron endowment in the vitamin D-deficient offspring in the absence of any observable increase in body weight or kidney size. As expected, there was a significant linear correlation between kidney weight and nephron number in the control group, but this association was not observed in the vitamin D-deficient offspring, implying that the regulation of nephrogenesis has been altered by vitamin D deficiency. However, since nephron numbers were counted when the rats were 7 weeks of age, we cannot exclude the possibility that there was a greater age-related loss of glomeruli in the control kidneys, since the completion of nephrogenesis at postnatal day 10. However, we consider an age-related loss in nephrons to have been minimal in both experimental groups, since, at 7 weeks of age, the rats were only approaching adolescence. Accompanying the increase in the number of nephrons in the kidneys of the vitamin D-deficient offspring there was a reduction in renal corpuscle size. Similar inverse correlations between nephron number and glomerular size have previously been described in experimental [23, 29] and human studies [18]. In light of our current findings, the question arises: How does vitamin D deficiency in the foetus lead to an increase in nephrogenesis in the developing kidney? Although this information cannot be derived from our findings, recent reports in the literature suggest two potential mechanisms. Firstly, there is experimental evidence to suggest that 1,25dihydroxyvitamin D3 (1,25(OH)2D3) acts as a negative regulator of renin gene expression in the kidney [35, 36] and this is independent of angiotensin II feedback regulation [37]. Hence, it may be upregulation of renal angiotensin II production in the vitamin D-deficient kidneys that leads to the rise in nephron number, since angiotensin II is linked to stimulation of nephrogenesis [38, 39]. Indeed, suppression of the renin–angiotensin system is thought to play a key role in the congenital nephron deficit associated with maternal protein restriction in newborn rats and to be linked to the programming of hypertension in this model [40]. Alternatively, it is well known that 1,25(OH)2D3 plays a key role in cell differentiation/maturation and is a potent inhibitor of cell proliferation [31]. Of particular relevance to our findings, these maturational and anti-proliferative effects of vitamin D have been reported in both the developing [41] and adult [42, 43] kidney. Hence, an alternative explanation for the increased nephron endowment in the vitamin Ddeficient offspring may be that nephrogenic proliferation is prolonged, without the appropriate switch to nephron maturation. If this is the case, it is likely that the nephrons, although more numerous, may not be fully matured and, hence, may be functionally impaired. Therefore, it is imperative that future studies gain an understanding of the mechanisms leading to enhanced nephrogenesis in the vitamin D-deficient foetus and also elucidate whether nephron function is normal or not. It is important to note that, in rodent transplantation studies of foetal kidneys into adult recipients, there was an increase in nephron number in the adult recipients if the metanephroi (immature kidneys) were pre-incubated with vitamin D3 or low concentrations of 25(OH)D3 [44]. The mechanisms leading to the stimulation of nephrogenesis under those conditions are currently unknown and will no doubt be different from those leading to stimulation of nephrogenesis in hypovitaminosis D in vivo described in our study. 60 In conclusion, the findings of this study demonstrate that vitamin D deficiency in early development can stimulate nephrogenesis. Whether the nephrons in these kidneys with supernumerary nephrons are functional, and thus confer an advantage to renal function, is yet to be elucidated. 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